Spectroscopic and functional characterization of a ligand coordination mutant of soybean lipoxygenase-1: First coordination sphere analogue of human 15-lipoxygenase

Lipoxygenases are an important class of non-heme iron enzymes which catalyz e the hydroperoxidation of unsaturated fatty acids. This study utilizes a c ombination of electron paramagnetic resonance (EPR), magnetic circular dich roism (MCD), and variable-temperature, variable-field (VTVH) MCD to probe t he ground state and excited states of the active site of an iron coordinati on mutant of soybean lipoxygenase-l (SLO-1), N694H, and relate the structur al modifications with kinetic changes; The sixth coordination ligand for SL O-1, Asn(694), is proposed to be a primary factor in the coordination flexi bility of the site and is reflected in both its ferrous MCD and ferric EPR spectroscopic properties. In the present study, we compare the spectroscopi c properties of the N694H SLO-1 mutant with those of wild-type (WT) SLO-1 a nd WT human 15-lipoxygenase (15-HLO) and determine that its properties are intermediate between the WT enzymes. WT 15-HLO has a His substitution in th e Asn694 equivalent position. The low-temperature MCD of the ferrous N694H SLO-1 shows that the site is 6-coordinate (6C), as in 15-HLO; however, the VTVH MCD is more similar to WT SLO-1, with a positive zero field splitting. The low-temperature EPR of ferric N694H SLO-1 exhibits a signal of interme diate rhombicity with an E/D of 0.08, which is intermediate between those f or WT SLO-1 (axial, E/D approximate to 0.01) and 15-HLO (rhombic, E/D appro ximate to 0.33). This intermediate spectroscopic behavior can be rationaliz ed by the stronger Ligation of the His compared to the Asn; however, since the site in WT SLO-1 is not optimized for His, it does not Ligate the iron as strongly as in the human enzyme. The H544N 15-HLO mutant was also expres sed; however, it does not bind iron for activity. The kinetics of N694H SLO -1 are also intermediate (k(cat) approximate to 10 +/- 2 s(-1)) between tho se for SLO-1 (k(cat) approximate to 280 +/- 8 s(-1)) and HLO (k(cat) approx imate to 6.2 +/- 0.1 s(-1)), which suggests that this ligand's interaction with the iron center may regulate the enzymatic activity. We propose that t his occurs through a change in reduction potential, which in turn affects t he hydrogen atom abstraction step in the reaction mechanism.